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bioRxiv · 10.1101/2025.07.22.665497

Endogenous gene editing of alveolar organoids reveals that expression of pathogenic variant SFTPC-I73T disrupts endosomal function, epithelial polarity and wound healing

Rutherford, E. N.·Sun, D.·Lim, K.·Edgar, J. R.·Matesic, L. E.·Marciniak, S. J.·Rawlins, E. L.·Dickens, J. A.
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Abstract

BackgroundIdiopathic pulmonary fibrosis is a fatal lung disease of progressive lung parenchymal scarring caused by the aberrant response of an alveolar epithelium repeatedly exposed to injury. Understanding epithelial dysfunction has been hampered by the lack of physiological alveolar type 2 (AT2) cell models and defined disease triggers. Monogenic forms of familial pulmonary fibrosis (FPF) caused by toxic gain-of-function variants provide an opportunity to investigate early pathogenic events. One such variant, surfactant protein C (SFTPC)-I73T, abnormally localises within AT2 cells and causes their dysfunction. MethodsWe used base editing of fetal lung-derived AT2 (fdAT2) organoids to create a heterozygous disease model of endogenous SFTPC-I73T expression. We also created an inducible overexpression system to interrogate temporal changes associated with SFTPC-I73T expression. We cultured fdAT2 both in 3D culture and at air-liquid interface to understand the importance of polarity cues and air exposure on disease phenotypes. ResultsIn our heterozygous endogenous expression system, we found that fdAT2 expressing SFTPC-I73T grew without a lumen and were unable to correctly polarise. SFTPC-I73T accumulated with time and caused gross enlargement of early endosomes, preventing correct apico-basal trafficking of multiple endosomally trafficked cargoes including polarity markers and cell adhesion proteins. This phenotype was exacerbated by air exposure and led to loss of epithelial monolayer integrity and abnormal wound healing after injury. ConclusionUsing endogenous gene editing for the first time in differentiated alveolar organoids, we have demonstrated that the pathogenic effects of SFTPC-I73T are mediated through endosomal dysfunction and abnormal epithelial organisation. This has important implications for AT2 function in vivo.

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Rutherford, E. N., Sun, D., Lim, K., Edgar, J. R., Matesic, L. E., Marciniak, S. J., Rawlins, E. L., Dickens, J. A.. 2025-07-22. Endogenous gene editing of alveolar organoids reveals that expression of pathogenic variant SFTPC-I73T disrupts endosomal function, epithelial polarity and wound healing. https://doi.org/10.1101/2025.07.22.665497

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